Thioflavin derivatives for use in the antemortem diagnosis of alzheimers disease and in vivo imaging and prevention of amyloid deposition

ABSTRACT

This invention relates to novel thioflavin derivatives, methods of using the derivatives in, for example, in vivo imaging of patients having neuritic plaques, pharmaceutical compositions comprising the thioflavin derivatives and method of synthesizing the compounds. The compounds find particular use in the diagnosis and treatment of patients having diseases where accumulation of neuritic plaques are prevalent. The disease states or maladies include but are not limited to Alzheimer&#39;s Disease, familial Alzheimer&#39;s Disease, Down&#39;s Syndrome and homozygotes for the apolipoprotein E4 allele.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Continuation of U.S. Ser. No. 10/859,600 filedJun. 3, 2004 which is a Continuation of Ser. No. 09/935,767 filed Aug.24, 2001 which claims priority from U.S. Provisional Application60/227,601, filed Aug. 24, 2000, incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to the identification of compounds thatare suitable for imaging amyloid deposits in living patients. Morespecifically, the present invention relates to a method of imagingamyloid deposits in brain in vivo to allow antemortem diagnosis ofAlzheimer's Disease. The present invention also relates to therapeuticuses for such compounds.

BACKGROUND OF THE INVENTION

Alzheimer's Disease (“AD”) is a neurodegenerative illness characterizedby memory loss and other cognitive deficits. McKhann et al., Neurology34: 939 (1984). It is the most common cause of dementia in the UnitedStates. AD can strike persons as young as 40-50 years of age, yet,because the presence of the disease is difficult to determine withoutdangerous brain biopsy, the time of onset is unknown. The prevalence ofAD increases with age, with estimates of the affected populationreaching as high as 40-50% by ages 85-90. Evans et al., JAMA 262: 2551(1989); Katzman, Neurology 43: 13 (1993).

In practice, AD is definitively diagnosed through examination of braintissue, usually at autopsy. Khachaturian, Arch. Neurol. 42: 1097 (1985);McKhann et al., Neurology 34: 939 (1984). Neuropathologically, thisdisease is characterized by the presence of neuritic plaques (NP),neurofibrillary tangles (NFT), and neuronal loss, along with a varietyof other findings. Mann, Mech. Ageing Dev. 31: 213 (1985). Post-mortemslices of brain tissue of victims of Alzheimer's disease exhibit thepresence of amyloid in the form of proteinaceous extracellular cores ofthe neuritic plaques that are characteristic of AD.

The amyloid cores of these neuritic plaques are composed of a proteincalled the β-amyloid (Aβ) that is arranged in a predominatelybeta-pleated sheet configuration. Mori et al., Journal of BiologicalChemistry 267: 17082 (1992); Kirschner et al., PNAS 83: 503 (1986).Neuritic plaques are an early and invariant aspect of the disease. Mannet al., J. Neurol. Sci. 89: 169; Mann, Mech. Ageing Dev. 31: 213 (1985);Terry et al., J. Neuropathol. Exp. Neurol 46: 262 (1987).

The initial deposition of Aβ probably occurs long before clinicalsymptoms are noticeable. The currently recommended “minimum microscopiccriteria” for the diagnosis of AD is based on the number of neuriticplaques found in brain. Khachaturian, Arch. Neurol., supra (1985).Unfortunately, assessment of neuritic plaque counts must be delayeduntil after death.

Amyloid-containing neuritic plaques are a prominent feature of selectiveareas of the brain in AD as well as Down's Syndrome and in personshomozygous for the apolipoprotein E4 allele who are very likely todevelop AD. Corder et al., Science 261: 921 (1993); Divry, P., J.Neurol. Psych. 27: 643-657 (1927); Wisniewski et al., in Zimmerman, H.M. (ed.): PROGRESS IN NEUROPATHOLOGY (Grune and Stratton, N.Y. 1973) pp.1-26. Brain amyloid is readily demonstrated by staining brain sectionswith thioflavin S or Congo red. Puchtler et al., J. Histochem. Cytochem.10: 35 (1962). Congo red stained amyloid is characterized by a dichroicappearance, exhibiting a yellow-green polarization color. The dichroicbinding is the result of the beta-pleated sheet structure of the amyloidproteins. Glenner, G. N. Eng. J. Med. 302: 1283 (1980). A detaileddiscussion of the biochemistry and histochemistry of amyloid can befound in Glenner, N. Eng. J. Med., 302: 1333 (1980).

Thus far, diagnosis of AD has been achieved mostly through clinicalcriteria evaluation, brain biopsies and post-mortem tissue studies.Research efforts to develop methods for diagnosing Alzheimer's diseasein vivo include (1) genetic testing, (2) immunoassay methods and (3)imaging techniques.

Evidence that abnormalities in Aβ metabolism are necessary andsufficient for the development of AD is based on the discovery of pointmutations in the Aβ precursor protein in several rare families with anautosomal dominant form of AD. Hardy, Nature Genetics 1: 233 (1992);Hardy et al., Science 256: 184 (1992). These mutations occur near the N-and C-terminal cleavage points necessary for the generation of Aβ fromits precursor protein. St. George-Hyslop et al., Science 235: 885(1987); Kang et al., Nature 325: 733 (1987); Potter WO 92/17152. Geneticanalysis of a large number of AD families has demonstrated, however,that AD is genetically heterogeneous. St. George-Hyslop et al., Nature347: 194 (1990). Linkage to chromosome 21 markers is shown in only somefamilies with early-onset AD and in no families with late-onset AD. Morerecently a gene on chromosome 14 whose product is predicted to containmultiple transmembrane domains and resembles an integral membraneprotein has been identified by Sherrington et al., Nature 375: 754-760(1995). This gene may account for up to 70% of early-onset autosomaldominant AD. Preliminary data suggests that this chromosome 14 mutationcauses an increase in the production of Aβ. Scheuner et al., Soc.Neurosci. Abstr. 21: 1500 (1995). A mutation on a very similar gene hasbeen identified on chromosome 1 in Volga German kindreds withearly-onset AD. Levy-Lahad et al., Science 269: 973-977 (1995).

Screening for apolipoprotein E genotype has been suggested as an aid inthe diagnosis of AD. Scott, Nature 366: 502 (1993); Roses, Ann. Neurol.38: 6-14 (1995). Difficulties arise with this technology, however,because the apolipoprotein E4 allele is only a risk factor for AD, not adisease marker. It is absent in many AD patients and present in manynon-demented elderly people. Bird, Ann. Neurol. 38: 2-4 (1995).

Immunoassay methods have been developed for detecting the presence ofneurochemical markers in AD patients and to detect an AD related amyloidprotein in cerebral spinal fluid. Warner, Anal. Chem. 59: 1203A (1987);World Patent No. 92/17152 by Potter; Glenner et al., U.S. Pat. No.4,666,829. These methods for diagnosing AD have not been proven todetect AD in all patients, particularly at early stages of the diseaseand are relatively invasive, requiring a spinal tap. Also, attempts havebeen made to develop monoclonal antibodies as probes for imaging of Aβ.Majocha et al., J. Nucl. Med., 33: 2184 (1992); Majocha et al., WO89/06242 and Majocha et al., U.S. Pat. No. 5,231,000. The majordisadvantage of antibody probes is the difficulty in getting these largemolecules across the blood-brain barrier. Using antibodies for in vivodiagnosis of AD would require marked abnormalities in the blood-brainbarrier in order to gain access into the brain. There is no convincingfunctional evidence that abnormalities in the blood-brain barrierreliably exist in AD. Kalaria, Cerebrovascular & Brain MetabolismReviews 4: 226 (1992).

Radiolabeled Aβ peptide has been used to label diffuse, compact andneuritic type plaques in sections of AD brain. See Maggio et al., WO93/04194. However, these peptides share all of the disadvantages ofantibodies. Specifically, peptides do not normally cross the blood-brainbarrier in amounts necessary for imaging and because these probes reactwith diffuse plaques, they may not be specific for AD.

The inability to assess amyloid deposition in AD until after deathimpedes the study of this devastating illness. A method of quantifyingamyloid deposition before death is needed both as a diagnostic tool inmild or clinically confusing cases as well as in monitoring theeffectiveness of therapies targeted at preventing Aβ deposition.Therefore, it remains of utmost importance to develop a safe andspecific method for diagnosing AD before death by imaging amyloid inbrain parenchyma in vivo. Even though various attempts have been made todiagnose AD in vivo, currently, there are no antemortem probes for brainamyloid. No method has utilized a high affinity probe for amyloid thathas low toxicity, can cross the blood-brain barrier, and binds moreeffectively to AD brain than to normal brain in order to identify ADamyloid deposits in brain before a patient's death. Thus, no in vivomethod for AD diagnosis has been demonstrated to meet these criteria.

Data suggest that amyloid-binding compounds will have therapeuticpotential in AD and type 2 diabetes mellitus. Morphological reactionsincluding, reactive astrocytosis, dystrophic neurites, activatedmicroglia cells, synapse loss, and full complement activation foundaround neuritic plaques all signify that neurotoxic and celldegenerative processes are occurring in the areas adjacent to these Aβdeposits. Joachim et al., Am. J. Pathol. 135: 309 (1989); Masliah etal., loc. cit. 137: 1293 (1990); Lue and Rogers, Dementia 3: 308 (1992).Aβ-induced neurotoxicity and cell degeneration has been reported in anumber of cell types in vitro. Yankner et al., Science 250: 279 (1990);Roher et al., BBRC 174: 572 (1991); Frautschy et al., Proc. Natl. Acad.Sci. 88: 83362 (1991); Shearman et al., loc. cit. 91: 1470 (1994). Ithas been shown that aggregation of the Aβ peptide is necessary for invitro neurotoxicity. Yankner, Neurobiol. Aging 13: 615 (1992). Recently,three laboratories have reported results which suggest that Congo redinhibits Aβ-induced neurotoxicity and cell degeneration in vitro.Burgevin et al., NeuroReport 5: 2429 (1994); Lorenzo and Yankner, Proc.Natl. Acad. Sci. 91: 12243 (1994); Pollack et al., Neuroscience Letters184: 113 (1995); Pollack et al., Neuroscience Letters 197: 211 (1995).The mechanism appears to involve both inhibition of fibril formation andprevention of the neurotoxic properties of formed fibrils. Lorenzo andYankner, Proc. Natl. Acad. Sci. 91: 12243 (1994). Congo red also hasbeen shown to protect pancreatic islet cells from the toxicity caused byamylin. Lorenzo and Yankner, Proc. Natl. Acad. Sci. 91: 12243 (1994).Amylin is a fibrillar peptide similar to Aβ which accumulates in thepancreas in type 2 diabetes mellitus.

It is known in the art that certain azo dyes, such as Congo red, may becarcinogenic. Morgan et al. Environmental Health Perspectives, 102(supp.) 2: 63-78, (1994). This potential carcinogenicity appears to bebased largely on the fact that azo dyes are extensively metabolized tothe free parent amine by intestinal bacteria. Cerniglia et al., Biochem.Biophys. Res. Com., 107: 1224-1229, (1982). In the case of benzidinedyes (and many other substituted benzidines), it is the free amine whichis the carcinogen. These facts have little implications for amyloidimaging studies in which an extremely minute amount of the high specificactivity radiolabelled dye would be directly injected into the bloodstream. In this case, the amount administered would be negligible andthe dye would by-pass the intestinal bacteria.

In the case of therapeutic usage, these facts have critical importance.Release of a known carcinogen from a therapeutic compound isunacceptable. A second problem with diazo dye metabolism is that much ofthe administered drug is metabolized by intestinal bacteria prior toabsorption. This lowered bioavailability remains a disadvantage even ifthe metabolites released are innocuous.

Thioflavin T is a basic dye first described as a selective amyloid dyein 1959 by Vassar and Culling (Arch. Pathol. 68: 487 (1959)). Schwartzet al. (Zbl. Path. 106: 320 (1964)) first demonstrated the use ofThioflavin S, an acidic dye, as an amyloid dye in 1964. The propertiesof both Thioflavin T and Thioflavin S have since been studied in detail.Kelenyi J. Histochem. Cytochem. 15: 172 (1967); Burns et al. J. Path.Bact. 94:337 (1967); Guntern et al. Experientia 48: 8 (1992); LeVineMeth. Enzymol. 309: 274 (1999). Thioflavin S is commonly used in thepost-mortem study of amyloid deposition in AD brain where it has beenshown to be one of the most sensitive techniques for demonstratingsenile plaques. Vallet et al. Acta Neuropathol. 83: 170 (1992).Thioflavin T has been frequently used as a reagent to study theaggregation of soluble amyloid proteins into beta-sheet fibrils. LeVineProt. Sci. 2: 404 (1993). Quaternary amine derivatives related toThioflavin T have been proposed as amyloid imaging agents, although noevidence of brain uptake of these agents has been presented. Caprathe etal. U.S. Pat. No. 6,001,331.

Thus, a need exists for amyloid binding compounds which enter the brainand bind selectively to amyloid.

A further need exists for amyloid binding compounds that are non-toxicand bioavailable and, consequently, can be used in therapeutics.

SUMMARY OF THE INVENTION

It is therefore one embodiment of the present invention to providecompounds which allow for a safe and specific method for diagnosing ADbefore death by in vivo imaging of amyloid in brain parenchyma.

It is another embodiment of the present invention to provide an approachfor identifying AD amyloid deposits in brain before a patient's death,using a high-affinity probe for amyloid which has low toxicity, cancross the blood-brain barrier, and can distinguish AD brain from normalbrain.

In accomplishing these and other embodiments of the invention, there isprovided, in accordance with one aspect of the invention, an amyloidbinding compound having one of structures A-E:

wherein Z is S, NR′, O or C(R′)₂ in which case the correct tautomericform of the heterocyclic ring becomes an indole in which R′ is H or alower alkyl group:

wherein Y is NR¹R², OR², or SR²;wherein the nitrogen of

is not a quaternary amine;or an amyloid binding compound having one of structures F-J or a watersoluble, non-toxic salt thereof:

wherein each Q is independently selected from one of the followingstructures:

wherein n=0, 1, 2, 3 or 4,

wherein Z is S, O, NR′, or C(R′)₂ in which R′ is H or a lower alkylgroup;wherein U is CR′ (in which R′ is H or a lower alkyl group) or N (exceptwhen U=N, then Q is not

wherein Y is NR¹R², OR², or SR²;wherein the nitrogen of

is not a quaternary amine;wherein each R¹ and R² independently is selected from the groupconsisting of H, a lower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or3), CF₃, CH₂—CH₂X, CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), (C═O)—R′,R_(ph), and (CH₂)_(n)R_(ph) (wherein n=1, 2, 3, or 4 and R_(ph)represents an unsubstituted or substituted phenyl group with the phenylsubstituents being chosen from any of the non-phenyl substituentsdefined below for R³-R¹⁴ and R′ is H or a lower alkyl group);and wherein each R³-R¹⁴ independently are selected from the groupconsisting of H, F, Cl, Br, I, a lower alkyl group, (CH₂)_(n)OR′(wherein n=1, 2, or 3), CF₃, CH₂—CH₂X, O—CH₂—CH₂X, CH₂—CH₂—CH₂X,O—CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), CN, (C═O)—R′, N(R′)₂, NO₂,(C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, R_(ph), CR′═CR′—R_(ph),CR₂′—CR₂′—R_(ph) (wherein R_(ph) represents an unsubstituted orsubstituted phenyl group with the phenyl substituents being chosen fromany of the non-phenyl substituents defined for R¹-R¹⁴ and wherein R′ isH or a lower alkyl group), a tri-alkyl tin and a chelating group (withor without a chelated metal group) of the form W-L or V—W-L, wherein Vis selected from the group consisting of —COO—, —CO—, —CH₂O— and—CH₂NH—; W is —(CH₂)_(n) where n=0, 1, 2, 3, 4, or 5; and L is:

wherein M is selected from the group consisting of Tc and Re;or wherein each R¹ and R² is a chelating group (with or without achelated metal group) of the form W-L, wherein W is —(CH₂)_(n) wheren=2, 3, 4, or 5; and L is:

wherein M is selected from the group consisting of Tc and Re;or wherein each R¹-R¹⁴ independently is selected from the groupconsisting of a chelating group (with or without a chelated metal ion)of the form W-L and V—W-L, wherein V is selected from the groupconsisting of —COO— and —CO—; W is —(CH₂)_(n) where n=0, 1, 2, 3, 4, or5; L is:

and wherein R¹⁵ independently is selected from one of:

or an amyloid binding, chelating compound (with or without a chelatedmetal group) or a water soluble, non-toxic salt thereof of the form:

wherein R¹⁵ independently is selected from one of:

and R¹⁶ is

wherein Q is independently selected from one of the followingstructures:

wherein n=0, 1, 2, 3 or 4,

wherein Z is S, NR′, O, or C(R′)₂ in which R′ is H or a lower alkylgroup;wherein U is N or CR′;wherein Y is NR¹R², OR²⁶, or SR²⁶;wherein each R¹⁷-R²⁴ independently is selected from the group consistingof H, F, Cl, Br, I, a lower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2,or 3), CF₃, CH₂—CH₂X, O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (whereinX=F, Cl, Br or I), CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′,SR′, COOR′, R_(ph), CR′═CR′—R_(ph) and CR₂′-CR₂′-R_(ph) (wherein R_(ph)represents an unsubstituted or substituted phenyl group with the phenylsubstituents being chosen from any of the non-phenyl substituentsdefined for R¹⁷-R²⁰ and wherein R′ is H or a lower alkyl group).

In a preferred embodiment, at least one of the substituents R¹-R¹⁴ ofthe structures A-E or F-J is selected from the group consisting of ¹³¹I,¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, CH₂—CH₂—X*, O—CH₂—CH₂—X*, CH₂—CH₂—CH₂—X*,O—CH₂—CH₂—CH₂—X* (wherein X*=¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br or ¹⁸F), ¹⁹F, ¹²⁵I,a carbon-containing substituent as specified above wherein at least onecarbon is ¹¹C or ¹³C and a chelating group (with chelated metal group)of the form W-L* or V—W-L*, wherein V is selected from the groupconsisting of —COO—, —CO—, —CH₂O— and —CH₂NH—; W is —(CH₂)_(n) wheren=0, 1, 2, 3, 4, or 5; and L* is:

wherein M* is ^(99m)Tc;

and a chelating group (with chelated metal group) of the form W-L* orV—W-L*, wherein V is selected from the group consisting of —COO—, —CO—,—CH₂O— and —CH₂NH—; W is —(CH₂)_(n) where n=0, 1, 2, 3, 4, or 5; and L*is:

and wherein R¹⁵ independently is selected from one of the following:

or the chelating compound (with chelated metal group) of the form:

wherein R¹⁵ independently is selected from one of the following:

wherein Q is independently selected from one of the followingstructures:

wherein n=0, 1, 2, 3 or 4,

wherein Z is S, NR′, O, or C(R′)₂ in which R′ is H or a lower alkylgroup;wherein U is N or CR′;wherein Y is NR¹R², OR², or SR²;

wherein each R¹⁷-R²⁴ independently is selected from the group consistingof H, F, Cl, Br, I, a lower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2,or 3), CF₃, CH₂—CH₂X, O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (whereinX=F, Cl, Br or I), CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′,SR′, COOR′, R_(ph), CR′═CR′-R_(ph) and CR₂′-CR₂′-R_(ph) (wherein R_(ph)represents an unsubstituted or substituted phenyl group with the phenylsubstituents being chosen from any of the non-phenyl substituentsdefined for R¹⁷-R²⁰ and wherein R′ is H or a lower alkyl group).

In another preferred embodiment, the thioflavin compounds are definedwhere Z=S, Y=N, R¹=H; and further wherein when the amyloid bindingcompound of the present invention is structure A or E, then R² isselected from the group consisting of a lower alkyl group, (CH₂)_(n)OR′(wherein n=1, 2, or 3), CF₃, CH₂—CH₂X, CH₂—CH₂—CH₂X (wherein X=F, Cl, Bror I), (C═O)—R′, R_(ph), and (CH₂)_(n)R_(ph) wherein n=1, 2, 3, or 4;

wherein when the amyloid binding compound of the present invention isstructure B, then R² is selected from the group consisting of(CH₂)_(n)OR′ (wherein n=1, 2, or 3, and where when R′=H or CH₃, n is not1). CF₃, CH₂—CH₂X and CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I);

wherein when the amyloid binding compound of the present invention isstructure C, then R² is selected from the group consisting of a loweralkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or 3, CF₃), CH₂—CH₂X,CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), (C═O)—H, R_(ph), and(CH₂)_(n)R_(ph) wherein n=1, 2, 3, or 4; or

wherein when the amyloid binding compound of the present invention isstructure D, then R² is selected from the group consisting of(CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X, CH₂—CH₂—CH₂X(wherein X=F, Cl, Br or I), (C═O)—R′, R_(ph), and CH₂R_(ph) wherein whenR² is (CH₂)_(n)R_(ph) R⁸ is not CH₃.

In another preferred embodiment, at least one of the substituents R³-R¹⁴of the amyloid binding compound of the present invention is selectedfrom the group consisting of ¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, CH₂—CH₂—X*,O—CH₂—CH₂—X*, CH₂—CH₂—CH₂—X*, O—CH₂—CH₂—CH₂—X* (wherein X*=¹³¹I, ¹²³I,⁷⁶Br, ⁷⁵Br or ¹⁸F), ¹⁹F, ¹²⁵I and a carbon-containing substituent asspecified in the definition of the compounds having one of thestructures A-E or F-J, wherein at least one carbon is ¹¹C or ¹³C, achelating group (with chelated metal group) of the form W-L* or V—W-L*,wherein V is selected from the group consisting of —COO—, —CO—, —CH₂O—and

—CH₂NH—; W is —(CH₂)_(n) where n=0, 1, 2, 3, 4, or 5; and L* is:

wherein M* is ^(99m)Tc;and a chelating group (with chelated metal group) of the form W-L* orV—W-L*, wherein V is selected from the group consisting of —COO—, —CO—,—CH₂O— and —CH₂NH—; W is—(CH₂)_(n) where n=0, 1, 2, 3, 4, or 5; and L* is:

and wherein R¹⁵ independently is selected from one of the following:

or the chelating compound (with chelated metal group) of the form:

wherein R¹⁵ independently is selected from one of the following:

wherein Q is independently selected from one of the followingstructures:

wherein n=0, 1, 2, 3 or 4,

wherein Z is S, NR′, O, or C(R′)₂ in which R′ is H or a lower alkylgroup;wherein U is N or CR′;wherein Y is NR¹R², OR², or SR²;

wherein each R¹⁷-R²⁴ independently is selected from the group consistingof H, F, Cl, Br, I, a lower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2,or 3), CF₃, CH₂—CH₂X, O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (whereinX=F, Cl, Br or I), CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′,SR′, COOR′, R_(ph), CR′═CR′-R_(ph) and CR₂′-CR₂′-R_(ph) (wherein R_(ph)represents an unsubstituted or substituted phenyl group with the phenylsubstituents being chosen from any of the non-phenyl substituentsdefined for R¹⁷-R²⁰ and wherein R′ is H or a lower alkyl group).

In especially preferred embodiments, the compound is selected fromstructures A-E, and Z=S, Y=N, R′=H, R¹=H, R²=CH₃ and R³-R¹⁴ are H;

Z=S, Y=O, R′=H, R²=CH₃ and R³-R¹⁴ are H; Z=S, Y=N, R′=H, R¹⁻⁴=H, R⁵=I,and R⁶-R¹⁴ are H; Z=S, Y=N, R′=H, R¹⁻⁴=H, R⁵=I, R=OH and R⁶-R⁷ andR⁹-R¹⁴ are H; Z=S, Y=N, R′=H, R¹=H, R²=CH₂—CH₂—CH₂—F and R³-R⁴ are H;Z=S, Y=O, R′=H, R²=CH₂—CH₂—F and R³-R¹⁴ are H; Z=S, Y=N, R′=H, R¹⁻⁷=H,R⁸=O—CH₂—CH₂—F and R⁹-R¹⁴ are H; or Z=S, Y=N, R′=H, R¹=CH₃, R²⁻⁷=H,R⁸=O—CH₂—CH₂—F and R⁹-R¹⁴ are H.

In especially preferred embodiments, the compound is selected fromstructures F-J, and Z=S, Y=N, R′=H, R¹=H, R²=CH₃ and R³-R¹⁴ are H;

Z=S, Y=O, R′=H, R²═CH₃ and R³-R¹⁴ are H; Z=S, Y=N, R′=H, R¹⁻⁴=H, R⁵=I,and R⁶-R¹⁴ are H; Z=S, Y=N, R′=H, R¹⁻⁴=H, R⁵=I, R⁸=OH and R⁶-R⁷ andR⁹-R¹⁴ are H; Z=S, Y=N, R′=H, R¹=H, R²=CH₂—CH₂—CH₂—F and R³-R¹⁴ are H;Z=S, Y=O, R′=H, R²═CH₂—CH₂—F and R³-R¹⁴ are H; Z=S, Y=N, R′=H, R¹⁻⁷=H,R⁸=O—CH₂—CH₂—F and R⁹-R¹⁴ are H; or Z=S, Y=N, R′=H, R¹=CH₃, R²⁻⁷=H,R⁸=O—CH₂—CH₂—F and R⁹-R¹⁴ are H.

In another preferred embodiment, at least one of the substituents R³-R¹⁴is selected from the group consisting of CN, OCH₃, OH and NH₂.

In still another preferred embodiment, the amyloid binding compound isselected from the group consisting of structure B, structure C andstructure D; wherein R¹=H, R²=CH₃ and R⁸ is selected from the groupconsisting of CN, CH₃, OH, OCH₃ and NH₂, in a preferred aspect of thisembodiment, R³-R⁷ and R⁹-R¹⁴ are H.

In still another embodiment, the amyloid binding compounds of thepresent invention bind to Aβ with a dissociation constant (K_(D))between 0.0001 and 10.0 μM when measured by binding to synthetic Aβpeptide or Alzheimer's Disease brain tissue.

Another embodiment of the invention relates to a method for synthesizingthe amyloid binding compounds of the present invention having at leastone of the substituents R¹-R¹⁴ selected from the group consisting of¹³¹I, ¹²⁵I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, and ¹⁹F, comprising the step oflabeling the amyloid binding compound wherein at least one of thesubstituents R¹-R¹⁴ is a tri-alkyl tin, by reaction of the compound witha ¹³¹I, ¹²⁵I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, or ¹⁹F containing substance.

Another embodiment of the invention relates to a method for synthesizingthe amyloid binding compounds of the present invention having at leastone of the substituents R³-R¹⁴ selected from the group consisting of¹³¹I, ¹²⁵I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, and ¹⁹F, comprising the step oflabeling the amyloid binding compound of structure A-E or F-J whereinZ=S, Y=N, R¹=H and at least one of the substituents R³-R¹⁴ is atri-alkyl tin, by reaction of the compound with a ¹³¹I, ¹²⁵I, ¹²³I,⁷⁶Br, ⁷⁵Br, ¹⁸F, or ¹⁹F containing substance.

A further embodiment of the present invention relates to apharmaceutical composition for in vivo imaging of amyloid deposits,comprising (a) an amyloid binding compound chosen from the structuresA-E or F-J, and (b) a pharmaceutically acceptable carrier. A preferredaspect of the embodiment relates to a pharmaceutical composition for invivo imaging of amyloid deposits, comprising (a) an amyloid bindingcompound chosen from the structures A-E or F-J wherein Z=S, Y=N, R¹=H,and (b) a pharmaceutically acceptable carrier.

In another embodiment of the invention is an in vivo method fordetecting amyloid deposits in a subject, comprising the steps of: (a)administering a detectable quantity of a pharmaceutical compositioncomprising the labeled amyloid binding compound, and detecting thebinding of the compound to amyloid deposit in the subject. In apreferred aspect of this embodiment, the amyloid deposit is located inthe brain of a subject. In a particularly preferred aspect of thisembodiment, the subject is suspected of having a disease or syndromeselected from the group consisting of Alzheimer's Disease, familialAlzheimer's Disease, Down's Syndrome and homozygotes for theapolipoprotein E4 allele. In another particularly preferred aspect ofthis embodiment, the detecting is selected from the group consisting ofgamma imaging, magnetic resonance imaging and magnetic resonancespectroscopy. In a preferred aspect of this embodiment, the gammaimaging is either PET or SPECT. In another preferred aspect of thisembodiment, the pharmaceutical composition is administered byintravenous injection. In another preferred aspect of this embodiment,the ratio of (i) binding of the compound to a brain area other than thecerebellum to (ii) binding of the compound to the cerebellum, in asubject, is compared to the ratio in a normal subject.

Another embodiment relates to a method of detecting amyloid deposits inbiopsy or post-mortem human or animal tissue comprising the steps of:(a) incubating formalin-fixed or fresh-frozen tissue with a solution ofan amyloid binding compound of the present invention to form a labeleddeposit and then, (b) detecting the labeled deposits. In a preferredaspect of this embodiment, the solution is composed of 25-100% ethanol,with the remainder of the solution being water, wherein the solution issaturated with an amyloid binding compound according to the presentinvention. In a particularly preferred aspect of this embodiment, thesolution is composed of an aqueous buffer (such as tris or phosphate)containing 0-50% ethanol, wherein the solution contains 0.0001 to 100 μMof an amyloid binding compound according to the present invention. In aparticularly preferred aspect of this embodiment, the detecting iseffected by microscopic techniques selected from the group consisting ofbright-field, fluorescence, laser-confocal, and cross-polarizationmicroscopy.

A further embodiment relates to a method of quantifying the amount ofamyloid in biopsy or post-mortem tissue comprising the steps of: a)incubating a radiolabeled derivative of an amyloid binding compound ofthe present invention with a homogenate of biopsy or post-mortem tissue,wherein at least one of the substituents R¹-R¹⁴ of the compound islabeled with a radiolabel selected from the group consisting of ¹²⁵I,³H, and a carbon-containing substituent as specified by the amyloidbinding compound structures A-E or F-J, wherein at least one carbon is¹⁴C, b) separating the tissue-bound from the tissue-unbound radiolabeledderivative of an amyloid binding compound of the present invention, c)quantifying the tissue-bound radiolabeled derivative of an amyloidbinding compound of the present invention, and d) converting the unitsof tissue-bound radiolabeled derivative of an amyloid binding compoundof the present invention to units of micrograms of amyloid per 100 mg oftissue by comparison with a standard.

In a preferred aspect of the above embodiment, the radiolabeledderivative of the amyloid binding compound of the present invention or awater soluble, non-toxic salt thereof is according to one of theformulae A-E below:

wherein Z is S, NR′, O or C(R′)₂ in which case the correct tautomericform of the heterocyclic ring becomes an indole in which R′ is H or alower alkyl group:

wherein Y is NR¹R², OR², or SR²;wherein the nitrogen of any

group is not a quaternary amine;or the radiolabeled derivative of the amyloid binding compound of thepresent invention or a water soluble, non-toxic salt thereof isaccording to one of the formulae F-J below:or

wherein each Q is independently selected from one of the followingstructures:

wherein n=0, 1, 2, 3 or 4,

wherein Z is S, NR′, O, or C(R′)₂ in which R′ is H or a lower alkylgroup;wherein U is CR′ (in which R′ is H or a lower alkyl group) or N (exceptwhen U=N, then Q is not

wherein Y is NR¹R², OR², or SR²;wherein the nitrogen of

is not a quaternary amine;wherein each R¹ and R² independently is selected from the groupconsisting of H, a lower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or3), CF₃, CH₂—CH₂X, CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), (C═O)—R′,R_(ph), and (CH₂)_(n)R_(ph) (wherein n=1, 2, 3, or 4 and R_(ph)represents an unsubstituted or substituted phenyl group with the phenylsubstituents being chosen from any of the non-phenyl substituentsdefined below for R³-R¹⁴ and R′ is H or a lower alkyl group); andwherein each R³-R¹⁴ independently is selected from the group consistingof H, F, Cl, Br, I, a lower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2,or 3), CF₃, CH₂—CH₂X, O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (whereinX=F, Cl, Br or I), CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′,SR′, COOR′, R_(ph), CR′═CR′-R_(ph), CR₂′-CR₂′-R_(ph) (wherein R_(ph)represents an unsubstituted or substituted phenyl group with the phenylsubstituents being chosen from any of the non-phenyl substituentsdefined for R¹-R¹⁴ and wherein R′ is H or a lower alkyl group), atri-alkyl tin and a chelating group (with or without a chelated metalgroup) of the form W-L or V—W-L, wherein V is selected from the groupconsisting of —COO—, —CO—, —CH₂O— and —CH₂NH—; W is —(CH₂)_(n) wheren=0, 1, 2, 3, 4, or 5; and L is:

wherein M is selected from the group consisting of Tc and Re;or wherein each R¹ and R² is a chelating group (with or without achelated metal group) of the form W-L, wherein W is —(CH₂)_(n) wheren=2, 3, 4, or 5; and L is:

wherein M is selected from the group consisting of Tc and Re;

-   -   or wherein each R¹-R¹⁴ independently is selected from the group        consisting of a chelating group (with or without a chelated        metal ion) of the form W-L and V—W-L, wherein V is selected from        the group consisting of —COO— and —CO—; W is —(CH₂)_(n) where        n=0, 1, 2, 3, 4, or 5; L is:

and wherein R¹⁵ independently is selected from one of the following:

or an amyloid binding, chelating compound (with or without a chelatedmetal group) or a water soluble, non-toxic salt thereof of the form:

wherein R¹⁵ independently is selected from the following:

and R¹⁶ is

wherein Q is independently selected from one of the followingstructures:

wherein n=0, 1, 2, 3 or 4,

wherein Z is S, NR′, O, or C(R′)₂ in which R′ is H or a lower alkylgroup;wherein U is N or CR′;wherein Y is NR¹R², OR², or SR²;wherein each R¹⁷-R²⁴ independently is selected from the group consistingof H, F, Cl, Br, I, a lower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2,or 3), CF₃, CH₂—CH₂X, O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (whereinX=F, Cl, Br or I), CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′,SR′, COOR′, R_(ph), CR′═CR′—R_(ph) and CR₂′-CR₂′-R_(ph) (wherein R_(ph)represents an unsubstituted or substituted phenyl group with the phenylsubstituents being chosen from any of the non-phenyl substituentsdefined for R¹⁷-R²⁰ and wherein R′ is H or a lower alkyl group).

Another embodiment relates to a method of distinguishing an Alzheimer'sdisease brain from a normal brain comprising the steps of: a) obtainingtissue from (i) the cerebellum and (ii) another area of the same brainother than the cerebellum, from normal subjects and from subjectssuspected of having Alzheimer's disease; b) incubating the tissues witha radiolabeled derivative of a thioflavin amyloid binding compoundaccording to the present invention so that amyloid in the tissue bindswith the radiolabeled derivative of an amyloid binding compound of thepresent invention; c) quantifying the amount of amyloid bound to theradiolabeled derivative of an amyloid binding compound of the presentinvention according to the above recited method; d) calculating theratio of the amount of amyloid in the area of the brain other than thecerebellum to the amount of amyloid in the cerebellum; e) comparing theratio for amount of amyloid in the tissue from normal subjects withratio for amount of amyloid in tissue from subjects suspected of havingAlzheimer's disease; and f) determining the presence of Alzheimer'sdisease if the ratio from the brain of a subject suspected of havingAlzheimer's disease is above 90% of the ratios obtained from the brainsof normal subjects.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification beconsidered as exemplary only, with the true scope and spirit of theinvention being indicated by the following claims. Additionally, alldocuments referred to herein are expressly incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Shows the structures of a Thioflavin S and Thioflavin T;

FIG. 2 Shows the structures of two thioflavin derivatives according tothe invention;

FIG. 3 Shows four serial sections of fluorescent dyed brain frontalcortex of an AD patient;

FIG. 4 Shows proposed sites of binding of Chrysamine G and Thioflavin Tin β-sheet fibrils;

FIG. 5 Shows competition assay using Chrysamine G, Thioflavin S andThioflavin T, and derivatives of the present invention (BTA-0, BTA-1 andBTA-2);

FIG. 6 Shows time course radioactivity in the frontal cortex of baboonsinjected with labeled BTA-1,6-Meo-BTA-1 and 6-Me-BTA-1; and

FIG. 7 Shows a transverse positron emission tomography image of twolevels of baboon brain following i.v. injection of [N-methyl-¹¹C]BTA-1.

FIG. 8 Shows post-mortem sections of human and transgenic mouse brainstained with a derivative of the present invention (BTA-1).

FIG. 9 Shows in vivo labeling of amyloid plaques and vascular amyloidstained by a derivative of the present invention (BTA-1) in livingtransgenic mice imaged with multiphoton microscopy.

DETAILED DESCRIPTION OF THE INVENTION

The present invention exploits the ability of Thioflavin compounds andradiolabeled derivatives thereof to cross the blood brain barrier invivo and bind to Aβ deposited in neuritic (but not diffuse) plaques, toAβ deposited in cerebrovascular amyloid, and to the amyloid consistingof the protein deposited in NFT. The present compounds arenon-quaternary amine derivatives of Thioflavin S and T which are knownto stain amyloid in tissue sections and bind to synthetic Aβ in vitro.Kelenyi J. Histochem. Cytochem. 15: 172 (1967); Burns et al. J. Path.Bact. 94:337 (1967); Guntern et al. Experientia 48: 8 (1992); LeVineMeth. Enzymol. 309: 274 (1999).

The thioflavin derivatives of the present invention have each of thefollowing characteristics: (1) specific binding to synthetic Aβ in vitroand (2) ability to cross a non-compromised blood brain barrier in vivo.

As used herein to describe the thioflavin derivatives, “lower alkyl” isbranched or straight chain C₁-C₈, preferably C₁-C₆ and most preferablyC₁-C₄ (e.g., methyl, ethyl, propyl or butyl). When R¹-R¹⁴ is defined as“tri-alkyl tin”, the moiety is a tri-C₁-C₈ alkyl Sn moiety, preferablytri-C₁-C₆ alkyl Sn moiety, most preferably tri-C₁-C₄ alkyl Sn moiety(e.g., methyl, ethyl, propyl or butyl).

The method of this invention determines the presence and location ofamyloid deposits in an organ or body area, preferably brain, of apatient. The present method comprises administration of a detectablequantity of a pharmaceutical composition containing an amyloid bindingcompound chosen from structures A-E or F-J, as defined above, called a“detectable compound,” or a pharmaceutically acceptable water-solublesalt thereof, to a patient. A “detectable quantity” means that theamount of the detectable compound that is administered is sufficient toenable detection of binding of the compound to amyloid. An “imagingeffective quantity” means that the amount of the detectable compoundthat is administered is sufficient to enable imaging of binding of thecompound to amyloid.

The invention employs amyloid probes which, in conjunction withnon-invasive neuroimaging techniques such as magnetic resonancespectroscopy (MRS) or imaging (MRI), or gamma imaging such as positronemission tomography (PET) or single-photon emission computed tomography(SPECT), are used to quantify amyloid deposition in vivo. The term “invivo imaging” refers to any method which permits the detection of alabeled thioflavin derivative which is chosen from structures A-E orF-J, as described above. For gamma imaging, the radiation emitted fromthe organ or area being examined is measured and expressed either astotal binding or as a ratio in which total binding in one tissue isnormalized to (for example, divided by) the total binding in anothertissue of the same subject during the same in vivo imaging procedure.Total binding in vivo is defined as the entire signal detected in atissue by an in vivo imaging technique without the need for correctionby a second injection of an identical quantity of labeled compound alongwith a large excess of unlabeled, but otherwise chemically identicalcompound. A “subject” is a mammal, preferably a human, and mostpreferably a human suspected of having dementia.

For purposes of in vivo imaging, the type of detection instrumentavailable is a major factor in selecting a given label. For instance,radioactive isotopes and ¹⁹F are particularly suitable for in vivoimaging in the methods of the present invention. The type of instrumentused will guide the selection of the radionuclide or stable isotope. Forinstance, the radionuclide chosen must have a type of decay detectableby a given type of instrument. Another consideration relates to thehalf-life of the radionuclide. The half-life should be long enough sothat it is still detectable at the time of maximum uptake by the target,but short enough so that the host does not sustain deleteriousradiation. The radiolabeled compounds of the invention can be detectedusing gamma imaging wherein emitted gamma irradiation of the appropriatewavelength is detected. Methods of gamma imaging include, but are notlimited to, SPECT and PET. Preferably, for SPECT detection, the chosenradiolabel will lack a particulate emission, but will produce a largenumber of photons in a 140-200 keV range. For PET detection, theradiolabel will be a positron-emitting radionuclide such as ¹⁹F whichwill annihilate to form two 511 keV gamma rays which will be detected bythe PET camera.

In the present invention, amyloid binding compounds/probes are madewhich are useful for in vivo imaging and quantification of amyloiddeposition. These compounds are to be used in conjunction withnon-invasive neuroimaging techniques such as magnetic resonancespectroscopy (MRS) or imaging (MRI), positron emission tomography (PET),and single-photon emission computed tomography (SPECT). In accordancewith this invention, the thioflavin derivatives may be labeled with ¹⁹For ¹³C for MRS/MRI by general organic chemistry techniques known to theart. See, e.g., March, J. ADVANCED ORGANIC CHEMISTRY: REACTIONS,MECHANISMS, AND STRUCTURE (3rd Edition, 1985), the contents of which arehereby incorporated by reference. The thioflavin derivatives also may beradiolabeled with ¹⁸F, ¹¹C, ⁷⁵Br, or ⁷⁶Br for PET by techniques wellknown in the art and are described by Fowler, J. and Wolf, A. inPOSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M., Mazziota,J., and Schelbert, H. eds.) 391-450 (Raven Press, NY 1986) the contentsof which are hereby incorporated by reference. The thioflavinderivatives also may be radiolabeled with ¹²³I for SPECT by any ofseveral techniques known to the art. See, e.g., Kulkarni, Int. J. Rad.Appl. & Inst. (Part B) 18: 647 (1991), the contents of which are herebyincorporated by reference. In addition, the thioflavin derivatives maybe labeled with any suitable radioactive iodine isotope, such as, butnot limited to ¹³¹I, ¹²⁵I, or ¹²³I, by iodination of a diazotized aminoderivative directly via a diazonium iodide, see Greenbaum, F. Am. J.Pharm. 108: 17 (1936), or by conversion of the unstable diazotized amineto the stable triazene, or by conversion of a non-radioactivehalogenated precursor to a stable tri-alkyl tin derivative which thencan be converted to the iodo compound by several methods well known tothe art. See, Satyamurthy and Barrio J. Org. Chem. 48: 4394 (1983),Goodman et al., J. Org. Chem. 49: 2322 (1984), and Mathis et al., J.Labell. Comp. and Radiopharm. 1994: 905; Chumpradit et al., J. Med.Chem. 34: 877 (1991); Zhuang et al., J. Med. Chem. 37: 1406 (1994);Chumpradit et al., J. Med. Chem. 37: 4245 (1994). For example, a stabletriazene or tri-alkyl tin derivative of thioflavin or its analogues isreacted with a halogenating agent containing ¹³¹I, ¹²⁵I, ¹²³I, ⁷⁶Br,⁷⁵Br, ¹⁸F or ¹⁹F. Thus, the stable tri-alkyl tin derivatives ofthioflavin and its analogues are novel precursors useful for thesynthesis of many of the radiolabeled compounds within the presentinvention. As such, these tri-alkyl tin derivatives are one embodimentof this invention.

The thioflavin derivatives also may be radiolabeled with known metalradiolabels, such as Technetium-99m (^(99m)Tc). Modification of thesubstituents to introduce ligands that bind such metal ions can beeffected without undue experimentation by one of ordinary skill in theradiolabeling art. The metal radiolabeled thioflavin derivative can thenbe used to detect amyloid deposits. Preparing radiolabeled derivativesof Tc^(99m) is well known in the art. See, for example, Zhuang et al.,“Neutral and stereospecific Tc-99m complexes:[99mTc]N-benzyl-3,4-di-(N-2-mercaptoethyl)-amino-pyrrolidines (P-BAT)”Nuclear Medicine & Biology 26(2):217-24, (1999); Oya et al., “Small andneutral Tc(v)O BAT, bisaminoethanethiol (N2S2) complexes for developingnew brain imaging agents” Nuclear Medicine & Biology 25(2):135-40,(1998); and Hom et al., “Technetium-99m-labeled receptor-specificsmall-molecule radiopharmaceuticals: recent developments and encouragingresults” Nuclear Medicine & Biology 24(6):485-98, (1997).

The methods of the present invention may use isotopes detectable bynuclear magnetic resonance spectroscopy for purposes of in vivo imagingand spectroscopy. Elements particularly useful in magnetic resonancespectroscopy include ¹⁹F and ¹³C.

Suitable radioisotopes for purposes of this invention includebeta-emitters, gamma-emitters, positron-emitters, and x-ray emitters.These radioisotopes include ¹³¹I, ¹²³I, ¹⁸F, ¹¹C, ⁷⁵Br, and ⁷⁶Br.Suitable stable isotopes for use in Magnetic Resonance Imaging (MRI) orSpectroscopy (MRS), according to this invention, include ¹⁹F and ¹³C.Suitable radioisotopes for in vitro quantification of amyloid inhomogenates of biopsy or post-mortem tissue include ¹²⁵I, ¹⁴C, and ³H.The preferred radiolabels are ¹¹C or ¹⁸F for use in PET in vivo imaging,¹²³I for use in SPECT imaging, ¹⁹F for MRS/MRI, and ³H or ¹⁴C for invitro studies. However, any conventional method for visualizingdiagnostic probes can be utilized in accordance with this invention.

The method may be used to diagnose AD in mild or clinically confusingcases. This technique would also allow longitudinal studies of amyloiddeposition in human populations at high risk for amyloid deposition suchas Down's syndrome, familial AD, and homozygotes for the apolipoproteinE4 allele. Corder et al., Science 261: 921 (1993). A method that allowsthe temporal sequence of amyloid deposition to be followed can determineif deposition occurs long before dementia begins or if deposition isunrelated to dementia. This method can be used to monitor theeffectiveness of therapies targeted at preventing amyloid deposition.

Generally, the dosage of the detectably labeled thioflavin derivativewill vary depending on considerations such as age, condition, sex, andextent of disease in the patient, contraindications, if any, concomitanttherapies and other variables, to be adjusted by a physician skilled inthe art. Dosage can vary from 0.001 μg/kg to 10 μg/kg, preferably 0.01μg/kg to 1.0 μg/kg.

Administration to the subject may be local or systemic and accomplishedintravenously, intraarterially, intrathecally (via the spinal fluid) orthe like. Administration may also be intradermal or intracavitary,depending upon the body site under examination. After a sufficient timehas elapsed for the compound to bind with the amyloid, for example 30minutes to 48 hours, the area of the subject under investigation isexamined by routine imaging techniques such as MRS/MRI, SPECT, planarscintillation imaging, PET, and any emerging imaging techniques, aswell. The exact protocol will necessarily vary depending upon factorsspecific to the patient, as noted above, and depending upon the bodysite under examination, method of administration and type of label used;the determination of specific procedures would be routine to the skilledartisan. For brain imaging, preferably, the amount (total or specificbinding) of the bound radioactively labeled thioflavin derivative oranalogue of the present invention is measured and compared (as a ratio)with the amount of labeled thioflavin derivative bound to the cerebellumof the patient. This ratio is then compared to the same ratio inage-matched normal brain.

The pharmaceutical compositions of the present invention areadvantageously administered in the form of injectable compositions, butmay also be formulated into well known drug delivery systems (e.g.,oral, rectal, parenteral (intravenous, intramuscular, or subcutaneous),intracisternal, intravaginal, intraperitoneal, local (powders, ointmentsor drops), or as a buccal or nasal spray). A typical composition forsuch purpose comprises a pharmaceutically acceptable carrier. Forinstance, the composition may contain about 10 mg of human serum albuminand from about 0.5 to 500 micrograms of the labeled thioflavinderivative per milliliter of phosphate buffer containing NaCl. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike, as described, for instance, in REMINGTON'S PHARMACEUTICALSCIENCES, 15th Ed. Easton: Mack Publishing Co. pp. 1405-1412 and1461-1487 (1975) and THE NATIONAL FORMULARY XIV., 14th Ed. Washington:American Pharmaceutical Association (1975), the contents of which arehereby incorporated by reference.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions,saline solutions, parenteral vehicles such as sodium chloride, Ringer'sdextrose, etc. Intravenous vehicles include fluid and nutrientreplenishers. Preservatives include antimicrobials, anti-oxidants,chelating agents and inert gases. The pH and exact concentration of thevarious components of the pharmaceutical composition are adjustedaccording to routine skills in the art. See, Goodman and Gilman's THEPHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th Ed.).

Particularly preferred pharmaceutical compositions of the presentinvention are those that, in addition to specifically binding amyloid invivo and capable of crossing the blood brain barrier, are also non-toxicat appropriate dosage levels and have a satisfactory duration of effect.

According to the present invention, a pharmaceutical compositioncomprising thioflavin amyloid binding compounds, is administered tosubjects in whom amyloid or amyloid fibril formation are anticipated. Inthe preferred embodiment, such subject is a human and includes, forinstance, those who are at risk of developing cerebral amyloid,including the elderly, nondemented population and patients havingamyloidosis associated diseases and Type 2 diabetes mellitus. The term“preventing” is intended to include the amelioration of celldegeneration and toxicity associated with fibril formation. By“amelioration” is meant the treatment or prevention of more severe formsof cell degeneration and toxicity in patients already manifesting signsof toxicity, such as dementia.

The pharmaceutical composition comprises thioflavin amyloid bindingcompounds described above and a pharmaceutically acceptable carrier. Inone embodiment, such pharmaceutical composition comprises serum albumin,thioflavin amyloid binding compounds and a phosphate buffer containingNaCl. Other pharmaceutically acceptable carriers include aqueoussolutions, non-toxic excipients, including salts, preservatives, buffersand the like, as described, for instance, in REMINGTON'S PHARMACEUTICALSCIENCES, 15th Ed., Easton: Mack Publishing Co., pp. 1405-1412 and1461-1487 (1975) and THE NATIONAL FORMULARY XIV., 14th Ed. Washington:American Pharmaceutical Association (1975), and the UNITED STATESPHARMACOPEIA XVIII. 18th Ed. Washington: American PharmaceuticalAssociation (1995), the contents of which are hereby incorporated byreference.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions,saline solutions, parenteral vehicles such as sodium chloride, Ringer'sdextrose, etc. Intravenous vehicles include fluid and nutrientreplenishers. Preservatives include antimicrobial, anti-oxidants,chelating agents and inert gases. The pH and exact concentration of thevarious components the pharmaceutical composition are adjusted accordingto routine skills in the art. See, Goodman and Gilman's THEPHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th Ed.).

According to the invention, the inventive pharmaceutical compositioncould be administered orally, in the form of a liquid or solid, orinjected intravenously or intramuscularly, in the form of a suspensionor solution. By the term “pharmaceutically effective amount” is meant anamount that prevents cell degeneration and toxicity associated withfibril formation. Such amount would necessarily vary depending upon theage, weight and condition of the patient and would be adjusted by thoseof ordinary skill in the art according to well-known protocols. In oneembodiment, a dosage would be between 0.1 and 100 mg/kg per day, ordivided into smaller dosages to be administered two to four times perday. Such a regimen would be continued on a daily basis for the life ofthe patient. Alternatively, the pharmaceutical composition could beadministered intramuscularly in doses of 0.1 to 100 mg/kg every one tosix weeks.

According to the aspect of the invention which relates to a method ofdetecting amyloid deposits in biopsy or post-mortem tissue, the methodinvolves incubating formalin-fixed tissue with a solution of athioflavin amyloid binding compound chosen from structures A-E or F-J,described above. Preferably, the solution is 25-100% ethanol, (with theremainder being water) saturated with a thioflavin amyloid bindingcompound according to the invention. Upon incubation, the compoundstains or labels the amyloid deposit in the tissue, and the stained orlabeled deposit can be detected or visualized by any standard method.Such detection means include microscopic techniques such asbright-field, fluorescence, laser-confocal and cross-polarizationmicroscopy.

The method of quantifying the amount of amyloid in biopsy or post-mortemtissue involves incubating a labeled derivative of thioflavin accordingto the present invention, or a water-soluble, non-toxic salt thereof,with homogenate of biopsy or post-mortem tissue. The tissue is obtainedand homogenized by methods well known in the art. The preferred label isa radiolabel, although other labels such as enzymes, chemiluminescentand immunofluorescent compounds are well known to skilled artisans. Thepreferred radiolabel is ¹²⁵I, ¹⁴C or ³H, the preferred label substituentof an amyloid binding compound chosen from structures A-E or F-J is atleast one of R³-R¹⁴. Tissue containing amyloid deposits will bind to thelabeled derivatives of the thioflavin amyloid binding compounds of thepresent invention. The bound tissue is then separated from the unboundtissue by any mechanism known to the skilled artisan, such as filtering.The bound tissue can then be quantified through any means known to theskilled artisan. The units of tissue-bound radiolabeled thioflavinderivative are then converted to units of micrograms of amyloid per 100mg of tissue by comparison to a standard curve generated by incubatingknown amounts of amyloid with the radiolabeled thioflavin derivative.

The method of distinguishing an Alzheimer's diseased brain from a normalbrain involves obtaining tissue from (i) the cerebellum and (ii) anotherarea of the same brain, other than the cerebellum, from normal subjectsand from subjects suspected of having Alzheimer's disease. Such tissuesare made into separate homogenates using methods well known to theskilled artisan, and then are incubated with a radiolabeled thioflavinamyloid binding compound. The amount of tissue which binds to theradiolabeled thioflavin amyloid binding compound is then calculated foreach tissue type (e.g. cerebellum, non-cerebellum, normal, abnormal) andthe ratio for the binding of non-cerebellum to cerebellum tissue iscalculated for tissue from normal and for tissue from patients suspectedof having Alzheimer's disease. These ratios are then compared. If theratio from the brain suspected of having Alzheimer's disease is above90% of the ratios obtained from normal brains, the diagnosis ofAlzheimer's disease is made. The normal ratios can be obtained frompreviously obtained data, or alternatively, can be recalculated at thesame time the suspected brain tissue is studied.

Molecular Modeling

Molecular modeling was done using the computer modeling programAlchemy2000 Tripost, Inc. St. Louis, Mo.) to generate the Aβ peptidechains in the anti-parallel beta-sheet conformation. Kirschner et al.,Proc. Natl. Acad. Sci. U.S.A. 83: 503 (1986). The amyloid peptides wereplaced in hairpin loops (Hilbich et al., J. Mol. Biol. 218: 149 (1991))and used without further structural refinement. The Aβ peptides werealigned so that alternate chains were spaced 4.76 Å apart,characteristic of beta-sheet fibrils. Kirschner, supra. Thioflavin Tderivatives were energy minimized and aligned with the fibril model tomaximize contact with Asp-23/Gln-15/His-13 of Aβ(1-42)

Characterization of Specific Binding to Aβ Synthetic Peptide: Affinity,Kinetics, Maximum Binding

The characteristics of thioflavin derivative binding were analyzed usingsynthetic Aβ(1-40) and 2-(4′-[¹C]methylamino-phenyl)-benzothiazole([N-methyl-¹¹C]BTA-1) in phosphate-buffered saline (pH 7.0) or glycinebuffer/20% ethanol (pH 8.0) as previously described for Chysamine-Gbinding. Klunk et al. Neurobiol. Aging 15: 691 (1994).

Amino acid sequence for Aβ(1-40) is as follows: 1 2 3 4 5 6 7 8 9 10 AspAla Glu Phe Arg His Asp Ser Gly Tyr 11 12 13 14 15 16 17 18 19 20 GluVal His His Gln Lys Leu Val Phe Phe 21 22 23 24 25 26 27 28 29 30 AlaGlu Asp Val Gly Ser Asn Lys Gly Ala 31 32 33 34 35 36 37 38 39 40 IleIle Gly Leu Met Val Gly Gly Val Val

Preparation of Thioflavin Derivatives for Tissue Staining

Both Thioflavin S (ThS) and Thioflavin T (ThT) were utilized aspharmacophores (see, e.g., FIG. 1). It is noted that both compoundscontain quaternary amines and are, therefore, quite hydrophilic as aresult.

[C-14]ThT was synthesized and used to determine relative lipophilicityby partitioning between octanol and phosphate-buffered saline. The logof the partition coefficient, log P_(oct), was found to be 0.57 for[C-14]ThT. It was determined that the quaternary amine renders ThT toopolar for use as an effective brain imaging agent. Based on the resultsof lipophilic Congo red derivatives (phenols uncharged at physiologicpH, but potentially ionizable with a pK_(a) of ˜8.5) (Klunk et al.WO09634853A1, WO09847969A1, WO09924394A2), the inventors removed themethyl group from the benzothiazole nitrogen for the ThT derivatives.The removal of the methyl moiety eliminated the charged quaternary aminefrom the heterocycle portion of the molecule, leaving an aromatic aminewhich typically have pK_(b) values ˜5.5. Shorthand nomenclature for theThT derivatives is used wherein the basic backbone is designated BTA(for BenzoThiazole-Aniline). Substituents on the benzothiazole ring areplaced before the ‘B’ and the number of methyl groups on the anilinenitrogen is placed after the ‘A’ (see, e.g., FIG. 2).

i. Preliminary Tissue Staining with ThT and Derivatives

ThT (see, e.g., FIG. 1) is a fluorescent dye that has been used as ahistological stain for amyloid (Burns et al., “The specificity of thestaining of amyloid deposits with thioflavine T” Journal of Pathology &Bacteriology 94:337-344; 1967.). ThT weakly stains plaques (see, e.g.,FIG. 3), tangles, neuropil threads and cerebrovascular amyloid (CVA) inAD brain. Preliminary tissue staining shows that both the primary amine2-(4′-aminophenyl)-6-methyl-benzothiazole (6-Me-BTA-0) and the tertiaryamine 2-(4′-dimethylaminophenyl)-6-methyl-benzothiazole (6-Me-BTA-2)also stain plaques and tangles in post-mortem AD brain (see, e.g., FIG.3). Experiments in which the concentrations of 6-Me-BTA-0 and 6-Me-BTA-2were progressively decreased showed that staining by both 6-Me-BTA-0 and6-Me-BTA-1 could still be detected with staining solutions containingonly 10 nM of the BTA compound. In contrast, BTP (2-phenylbenzothiazole)does not appear to stain plaques, however, this compound is not nearlyas fluorescent as the BTA derivatives. Thus, in the development of thesecompounds, tissue staining has served the dual purpose of assessingspecificity of staining in AD brain tissue as well as assessing bindingaffinity by screening staining solutions over a range of concentrationssimilar to that employed in the binding assays.

ii. Binding Models of Congo Red Derivatives and ThT to Aβ

There are some theories about the binding mechanism of ThT to β-amyloid,but no specific theory has been proven or accepted. However, themechanism appears to be specific and saturable (LeVine, “Quantificationof beta-sheet amyloid fibril structures with thioflavin T” Meth.Enzymol. 309:272-284; 1999). Thus, it should be possible to localize thepotential binding site(s) on Aβ and develop a binding model in a manneranalogous to that used to develop the Congo red (CR)/Chrysamine-G (CG)binding model (Klunk et al., “Developments of small molecule probes forthe beta-amyloid protein of Alzheimer's disease” Neurobiol. Aging15:691-698; 1994.) based on the following structural and bindingproperties. First, ThT and CG have opposite charges at physiological pH,and it is unlikely that they share a common binding site. This issupported by the lack of competition of ThT for [³H]CG binding to Aβfibrils (see, e.g., FIG. 5).

Previous structural studies of Aβ fibrils (Hilbich et al., “Aggregationand secondary structure of synthetic amyloid beta A4 peptides ofAlzheimer's disease” Journal of Molecular Biology 218:149-63; 1991.) andCR and CG binding to Aβ fibrils suggested a molecular model in which CGbinds through a combination of electrostatic and hydrophobic interactionto the area of Lys-16 (see, e.g., FIG. 4). The studies of LeVine (LeVineibid) help localize the site of ThT binding to Aβ by showing that ThTbinds well to Aβ12-28, but negligibly to Aβ25-35. This suggests the ThTbinding site lies somewhere between residues 12 and 24 of Aβ. It islikely that the positively charged ThT (a quaternary amine) will beattracted to negatively charged (acidic) residues on Aβ. Between aminoacids 12 and 24, the only acidic residues are Glu-22 and Asp-23. Whileboth of these are candidates, the existing model predicts that Glu-22 isinvolved very near the Lys-16 binding site for CG. The current “working”model localizes ThT binding to the area of Asp-23—on the opposite sideof the fibril from the proposed CG site. Since the key feature of ThT(and CG) binding is the presence of a beta-sheet fibril, binding mustrequire more than just a single amino acid residue. The binding siteexists when residues not normally interacting in monomers are broughttogether in the beta-sheet fibril. Therefore, without being bound to anyone theory, it is believed that ThT also interacts via hydrogen bonds toHis-13 and Gln-15 of a separate, adjacent Aβ molecule comprising thebeta-sheet fibril.

iii. Radiolabeling of ThT and Radioligand Binding Assays

Assessing binding by tissue staining is useful, particularly forassessing specificity. The compound BTP, which is not very fluorescent,may not show staining either because it does not bind well enough, orbecause it is not fluorescent enough. In addition to the AD tissuestaining, quantitative binding assays can be conductedspectrophotometrically (LeVine ibid). This assay depends onmetachromatic spectral shift which occurs when ThT binds to the amyloidfibril. While this assay can be useful to individually screen highlyfluorescent compounds that show this metachromatic shift, it has notbeen determined to be useful for competition assays. For example, it iscommonly observed that test compounds (e.g., CG) quench the fluorescenceof the ThT-Aβ complex (as well as ThT alone). Compounds that quench, butdo not bind to the ThT site, will falsely appear to bind. Therefore, itis preferable to use radiolabeled ThT in typical radioligand bindingassays with aggregated Aβ. In this assay, inhibition of radiolabeled ThTbinding to Aβ trapped on filters would represent true inhibition of ThTbinding and does not require the test compound to be highly fluorescent.

The following examples are given to illustrate the present invention. Itshould be understood, however, that the invention is not to be limitedto the specific conditions or details described in these examples.Throughout the specification, any and all references to a publiclyavailable document, including U.S. patents, are specificallyincorporated into this patent application by reference.

EXAMPLES

All of the reagents used in the synthesis were purchased from AldrichChemical Company and used without further purification. Melting pointswere determined on MeI-TEMP II and were uncorrected. The ¹H NMR spectraof all compounds were measured on Bruker 300 using TMS as internalreference and were in agreement with the assigned structures. The TLCwas performed using Silica Gel 60 F₂₅₄ from EM Sciences and detectedunder UV lamp. Flash chromatography was performed on silica gel 60(230-400 mesh. purchased from Mallinckrodt Company. The reverse phaseTLC were purchased from Whiteman Company.

SYNTHESIS EXAMPLES Example 1 Synthesis of Primuline Base Derivatives

Route 1: Example of the synthesis of Primuline compounds is according tothe reaction scheme shown below:

The primuline derivatives are prepared based on Schubert's method(Schubert, M. Zur Kenntnis der Dehydrothiotoluidin- andPrimulin-sulfosäuren, Justus Liebigs Ann. Chem. 558, 10-33, 1947)through condensation of 2-amino-5-methylthiophenol with2-(p-nitrophenyl)-benzothiazole-6-carboxylic chloride and subsequentreduction of the nitro group with tin chloride in ethanol. Substitutedderivatives of primuline base are synthesized with the appropriatesubstituted p-nitrobenzoylchlorides and R⁷-R¹⁰ substituted2-aminothiophenol.

Following the same strategy as above, the other claimed primulinderivatives may be synthesized by substituting the appropriatesubstituted 3-mercapto-4-aminobenzoic acid derivative (e.g. 2-, 5-, or6-methyl-3-mercapto-4-aminobenzoic acid), the appropriate4-nitro-benzoyl chloride derivative (e.g. 2- or 3-methyl-4-nitro-benzoylchloride) or the appropriate 2-amino-5-methylthiophenol derivative (e.g.3,5-, 4,5-, or 5,6-dimethyl-2-aminothiophenol).

Example 2 Synthesis of 2-[2-(4′-aminophenyl)-ethylenyl)-benzothiazolederivatives

Route 3: Example of the synthesis of BTEA-0, 1, 2 and BTAA-0, 1, 2,which are representative of the group of BTEA and BTAA compounds wasaccording to the reaction scheme shown below:

(a) Trans-2-(4-Nitrophenylethenyl)benzothiazole (11)

trans-4-Nitrocinnamyl chloride 10 (1.77 g, 9.5 mmol, 1.2 eq.) in DMF (20ml) was added dropwise to a solution of 2-aminothiophenol 9 (1.0 g, 8.0mmol) in DMF (15 ml) at room temperature. The reaction mixture wasstirred at room temperature for overnight. The reaction mixture waspoured into a solution of 10% sodium carbonate (100 ml). The participatewas collected by filtration under reduced pressure. Recrystallizationfrom methanol gave 1.92 g (85.1%) of the product 11.

(b) 2-(4-Aminophenylethenyl)benzothiazole (12)

A mixture of 2-(4-nitrophenylethenyl)benthiazole 11 (500 mg, 1.7 mmol)and tin(II) chloride dihydrate (1.18 g, 5.2 mmol) in anhydrous ethanol(20 ml) was refluxed under N2 for 4 hrs. Ethanol was removed by vacuumevaporation. The residue was dissolved into ethyl acetate (20 ml),washed with NaOH solution (1 N, 3×20 ml) and water (3×20 ml), and driedover MgSO₄. Evaporation to dryness gave 40 mg (8.0%) of product 12.

(c) 2-(4-Methylminophenylethenyl)benzothiazole (13)

A mixture of 2-(4-aminophenylethenyl)benzothiazole 12 (7 mg), MeI (3.9mg) and anhydrous K₂CO₃ (100 mg) in DMSO (anhydrous, 0.5 ml) was heatedat 100° C. for 16 hrs. The reaction mixture was purified with reversephase TLC (MeOH:H2O=7:1) to give 2.5 mg (32.7%) of the product 13.

(d) 2-(4-aminophenylethylene)benzothiazole (14)

2-(4-Nitrophenylethenyl)benzothiazole (30 mg, 0.10 mmol) was dissolvedin MeOH (10 mL). Pd/C (10%, 40 mg) was added and the reaction mixturewas stirred under H₂ atmosphere at room temperature 60 hrs. The catalystwas filtrated and washed with methanol (ca. 2 ml). Evaporation of thefiltrate gave the crude product which was purified with TLC(hexanes:ethyl acetate=70:40,) to give 15 mg (50%) of the product. ¹HNMR(300 MHz, MeOH-d₄) δ: 7.88 (d, J=8.3 Hz, 1H, H-7), 7.86 (d, J=8.1 Hz,1H, H-4), 7.48 (dd, J₁=J₂=6.2 Hz, 1H, H-5 or H-6), 7.38 (dd, J₁=J₂=8.2Hz, 1H, H-5 or H-6), 6.96 (d, J=6.8 Hz, 2H, H-2′,6′), 6.62 (d, J=6.8 Hz,2H, H-3′, 5′), 3.36 (t, J=7.4 Hz, 2H, CH₂), 3.03 (t, J=7.4 Hz, 2H, CH₂).

(e) 2-(4-Dimethylaminophenylethenyl)benzothiazole (16)

A mixture of 2-aminothiophenol 9 (0.51 g, 4.1 mmol)trans-4-dimethylaminocinnamic acid 14 (0.79 g, 4.1 mmol) and PPA (10 g)was heated to 220° C. for 4 hrs. The reaction mixture was cooled to roomtemperature and poured into 10% of potassium carbonate solution (˜400mL). The residue was collected by filtration under reduced pressure.Purification with flush column (hexanes:ethyl acetate=2:1) gave 560 mg(48.7%) of product 15 as a yellow solid.

(f) 2-(4-Dimethylaminophenylethylene)benzothiazole (17)

2-(4-Dimethylaminophenylethenyl)benzothiazole (12 mg, 0.038 mmol) wasdissolved in MeOH (5 mL). Pd/C (10%, 20 mg) was added and the reactionmixture was stirred under H₂ atmosphere at room temperature 16 hr. Thecatalyst was filtrated and washed with methanol (ca. 1 ml). Evaporationof the filtrate gave 7 mg (58%) of the product. ¹HNMR (300 MHz,Acetone-d₆) δ: 7.97 (d, J=8.3 Hz, 1H, H-7), 7.93 (d, J=8.1 Hz, 1H, H-4),7.48 (dt, J=6.2 Hz, J=1.1 Hz 1H, H-5 or H-6), 7.38 (dt, J=8.2 Hz, J=1.1Hz, 1H, H-5 or H-6), 7.13 (d, J=6.8 Hz, 2H, H-2′,6′), 6.68 (d, J=6.8 Hz,2H, H-3′, 5′), 3.37 (t, J=7.4 Hz, 2H, CH₂), 3.09 (t, J=7.4 Hz, 2H, CH₂),2.88 (s, 6H, NMe₂).

Example 3 Synthesis of 2-(4′-aminophenyl)-benzothiazole derivatives

Route 1: Example of the synthesis of 6-MeO-BTA-0, -1, -2, which arerepresentative of the group of BTA compounds with substituents R₇-R₁₀ aswell as R₃-R₆ (Shi et al., “Antitumor Benzothiazoles. 3. Synthesis of2-(4-Aminophenyl)benzothiazoles and Evaluation of Their Activitiesagainst Breast Cancer Cell Lines in Vitro and in Vivo” J. Med. Chem.39:3375-3384, 1996):

(a) 4-Methoxy-4′-nitrobenzanilide (3)

p-Anisidine 1 (1.0 g, 8.1 mmol) was dissolved in anhydrous pyridine (15ml), 4-nitrobenzoyl chloride 2 (1.5 g, 8.1 mmol) was added. The reactionmixture was allowed to stand at room temperature for 16 hrs. Thereaction mixture was poured into water and the precipitate was collectedwith filtrate under vacuum pressure and washed with 5% sodiumbicarbonate (2×10 ml). The product 3 was used in the next step withoutfurther purification. ¹HNMR (300 MHz, DMSO-d₆) δ: 10.46 (s, 1H, NH),8.37 (d, J=5.5 Hz, 2H, H-3′,5′), 8.17 (d, J=6.3 Hz, 2H, H-2′,6′), 7.48(d, J=6.6 Hz, 2H), 6.97 (d, J=6.5 Hz, 2H), 3.75 (s, 3H, MeO).

(b) 4-Methoxy-4′-nitrothiobenzanilide (4)

A mixture of 4-methoxy-4′-nitrothiobenzaniline 3 (1.0 g, 3.7 mmol) andLawesson's reagent (0.89 g, 2.2 mmol, 0.6 equiv.) in chlorobenzene (15mL) was heated to reflux for 4 hrs. The solvent was evaporated and theresidue was purified with flush column (hexane:ethyl acetate=4:1) togive 820 mg (77.4%) of the product 4 as orange color solid. ¹HNMR (300MHz, DMSO-d₆) δ: 8.29 (d, 2H, H-3′,5′), 8.00 (d, J=8.5 Hz, 2H, H-2′,6′),7.76 (d, 2H), 7.03 (d, J=8.4 Hz, 2H), 3.808.37 (d, J=5.5 Hz, 2H,H-3′,5′), 8.17 (d, J=6.3 Hz, 2H, H-2′,6′), 7.48 (d, J=6.6 Hz, 2H), 6.97(d, J=6.5 Hz, 2H), 3.75 (s, 3H, MeO). (s, 3H, MeO).

(c) 6-Methoxy-2-(4-nitrophenyl)benzothiazole (5)

4-Methoxy-4′-nitrothiobenzanilides 4 (0.5 g, 1.74 mmol) was wetted witha little ethanol (˜0.5 mL), and 30% aqueous sodium hydroxide solution(556 mg 13.9 mmol. 8 equiv.) was added. The mixture was diluted withwater to provide a final solution/suspension of 10% aqueous sodiumhydroxide. Aliquots of this mixture were added at 1 min intervals to astirred solution of potassium ferricyanide (2.29 g, 6.9 mmol, 4 equiv.)in water (5 mL) at 80-90° C. The reaction mixture was heated for afurther 0.5 h and then allowed to cool. The participate was collected byfiltration under vacuum pressure and washed with water, purified withflush column (hexane:ethyl acetate=4:1) to give 130 mg (26%) of theproduct 5. ¹HNMR (300 MHz, Acetone-d₆) δ: 8.45 (m, 4H), 8.07 (d, J=8.5Hz, 1H, H-4), 7.69 (s, 1H, H-7), 7.22 (d, J=9.0 Hz, 1H, H-5), 3.90 (s,3H, MeO)

(d) 6-Methoxy-2-(4-aminophenyl)benzothiazole (6)

A mixture of the 6-methoxy-2-(4-nitrophenyl)benzothiazoles 5 (22 mg,0.077 mmol) and tin(II) chloride dihydrate (132 mg, 0.45 mmol) inboiling ethanol was stirred under nitrogen for 4 hrs. Ethanol wasevaporated and the residue was dissolved in ethyl acetate (10 mL),washed with 1 N sodium hydroxide (2 mL) and water (5 mL), and dried overMgSO₄. Evaporation of the solvent gave 19 mg (97%) of the product 6 asyellow solid.

(e) 6-Methoxy-2-(4-methylaminophenyl)benzothiazole (7) and6-Methoxy-2-(4-dimethylaminophenyl)benzothiazole (8)

A mixture of 6-methoxy-2-(4-aminophenyl)benzothiazole 6 (15 mg, 0.059mmol), MeI (8.3 mg, 0.060 mmol) and K₂CO₃ (100 mg, 0.72 mmol) in DMSO(anhydrous, 0.5 ml) was heated at 100° C. for 16 hrs. The reactionmixture was purified by reverse phase TLC (MeOH:H2O=7:1) to give 2.0 mg(13.3%) of 6-methoxy-2-4-methylaminophenylbenzothiazole 7 and 6 mg (40%)of 6-methoxy-2-(4-dimethylaminophenyl)benzothiazole 8. ¹HNMR of 7 (300MHz, Acetone-d₆) δ: 7.85 (d, J=8.7 Hz, 2H, H-2′ 6′), 7.75 (dd, J=8.8 Hz,J=1.3 Hz, 1H, H-4), 7.49 (d, J=2.4 Hz, 1H, H-7), 7.01 (dd, J=8.8 Hz,J=2.4 Hz, H-5), 6.78 (d, J=7.6 Hz, 2H, H-3′ 5′), 3.84 (s, 3H, MeO), 2.91(s, 3H, NMe), ¹HNMR of 8 (300 MHz, Acetone-d₆) δ: 7.85 (d, J=8.7 Hz, 2H,H-2′ 6′), 7.75 (dd, J=8.8 Hz, J=1.3 Hz, 1H, H-4), 7.49 (d, J=2.4 Hz, 1H,H-7), 7.01 (dd, J=8.8 Hz, J=2.4 Hz, H-5), 6.78 (d, J=7.6 Hz, 2H, H-3′5′), 3.84 (s, 3H, MeO), 3.01 (s, 6H, NMe₂),

Following the same strategy as above, the other claimed2-(4′-aminophenyl)-benzothiazole derivatives may be synthesized bysubstituting the appropriate substituted aniline derivative (e.g. 2-,3-, or 4-methylaniline) and the appropriate 4-nitro-benzoyl chloridederivative (e.g. 2- or 3-methyl-4-nitro-benzoyl chloride).

Example 4 Synthesis of BTA Derivatives without R⁷-R¹⁰ Substitution

Route 2: Example of the synthesis of BTA-0, -1, -2 compounds, which arerepresentative of the group of BTA compounds without R⁷-R¹⁰ (Garmaise etal., “Anthelmintic Quaternary Salts. III. Benzothiazolium Salts” J. Med.Chem. 12:30-36 1969):

(a) 2-(4-Nitrophenyl)benzothiazole (19)

A solution of 4-nitrobenzoyl chloride (1.49 g, 8.0 mmol) in benzeneanhydrous, 10 mL) was added dropwise to 2-aminothiophenol (1.0 g, 8.0mmol in 10 ml of benzene) at room temperature. The reaction mixture wasallowed to stir for 16 hr. The reaction was quenched with water (20 mL).The aqueous layer was separated and extracted with ethyl acetate (3×10ml). The combined organic layers were dried and evaporated. The crudeproduct was purified with flush column, (hexane:ethyl acetate=85:15) togive 1.5 g (73.2%) of product as light yellow solid.

(b) 2-(4-Aminophenyl)benzothiazole (20)

A mixture of 2-(4-nitrophenyl)benzothiazole (105 mg, 0.40 mmol) andtin(II) chloride dihydrate (205 mg, 0.91 mmol) in ethanol (20 mL) wasrefluxed under N2 for 4 hrs. After removing ethanol by vacuumevaporation. The residue was dissolved into ethyl acetate (20 ml), andwashed with NaOH solution (1 N, 3×20 ml) and water (3×20 ml), dried andevaporated to dryness to give 102 mg (97%) of the product

(c) 2-(4-Methylaminophenyl)benzothiazole (21) and2-(4-dimethylaminophenyl)benzothiazole (23)

A mixture of 2-(4-aminophenyl)benzothiazole 20 (15 mg, 0.066 mmol), MeI(9.4 mg, 0.066 mg) and K₂CO₃ (135 mg, 0.81 mmol) in DMSO (anhydrous, 0.5ml) was heated at 100° C. for 16 hrs. The reaction mixture was purifiedby reverse phase TLC (MeOH:H2O=6:1) to give 1.5 mg (10%) of2-(4-methylminophenyl)benzothiazole 21 and 2.5 mg (16.7%) of2-(4-dimethylaminophenyl)benzothiazole 23.

(d) 2-(4-Dimethylaminophenyl)benzothiazole (23)

The mixture of 2-aminothiophenol 9 (0.5 g, 4.0 mmol)4-dimethylaminobenzoic acid 22 (0.66 g, 4.0 mmol) and PPA (10 g) washeated to 220° C. for 4 hrs. The reaction mixture was cooled to roomtemperature and poured into a solution of 10% potassium carbonate (˜400mL). The residue was collected by filtration under vacuum pressure togive 964 mg of the product 23, which was ca. 90% pure based on the ¹HNMRanalysis. Recrystallization of 100 mg of 23 in MeOH gave 80 mg of thepure product. ¹HNMR (300 MHz, Acetone-d₆) δ: 7.12 (d, J=7.7 Hz, 1H,H-7), 7.01 (d, J=9.0 Hz, 1H, H-4), 6.98 (d, J=9.1 Hz, 2H, H-2′,6′), 6.56(t, J=7.8 Hz, J=7.3 Hz, 1H, H-5 or H-6), 5.92 (d, J=8.9 Hz, 1H,H-3′,5′), 2.50 (s, 6H, NMe₂).

Following the same strategy as above, the other claimed2-(4′-aminophenyl)-benzothiazole derivatives may be synthesized bysubstituting appropriate 4-nitro-benzoyl chloride derivative (e.g. 2- or3-methyl-4-nitro-benzoyl chloride) or appropriate4-dimethylamino-benzoic acid derivative (e.g. 2- or3-methyl-4-dimethylamino-benzoic acid).

Example 5 Synthesis of bis-2,2′-(4′-aminophenyl)-dibenzothiazolederivatives

Route 1: Following the general procedure for 6-MeO-BTA compoundsdescribed above but substituting benzidine for p-anisidine and using 16equivalents of 4-nitrobenzoyl chloride results in the followingcompound:

Following the same strategy as above, the otherbis-2,2′-(4′-aminophenyl)-dibenzothiazole derivatives may be synthesizedvia the appropriate substituted benzidine derivative (e.g. 2,2′-,3,3′-dimethylbenzidine) and the appropriate 4-nitro-benzoyl chloridederivative (e.g. 2- or 3-methyl-4-nitro-benzoyl chloride).

Route 2: The unsymmetric bis-2,2′-(4′-aminophenyl)-dibenzothiazolederivatives are synthesized through palladium catalyzed Suzuki couplingof the appropriate substituted 6-iodo-(2-p-nitrophenyl)benzothiazoles,which can be prepared following the same strategy as 6-MeO-BTA compoundsand subsequent reduction of nitro groups (Ishiyama et al., “Palladium(0)-Catalyzed Cross-Coupling Reaction of Alkoxydiboron with Haloarenes:A Direct Procedure for Arylboronic Esters” Tetrahedron Lett., 38, 3447,1997).

BIOLOGICAL EXAMPLES Example 6 Determination of Affinity for Aβ and BrainUptake of Thioflavin Derivatives

Initial competitive binding studies using [³H]CG and synthetic Aβ(1-40)were conducted to determine if CG, ThS and ThT bound to the samesite(s). It has been determined that ThS competed with [³H]CG forbinding sites on Aβ(1-40), but ThT did not (see, e.g., FIG. 5). Highspecific activity [N-methyl-¹¹C]BTA-1 (see Table 1) was then synthesizedby methylation of BTA-0. Bindings studies were performed with[N-methyl-¹¹C]BTA-1 and 200 nM Aβ(1-40) fibrils. The specific binding of[N-methyl-¹¹C]BTA-1 was ˜70%. FIG. 5 (see the right panel) showscompetition curves for Aβ sites by ThT, BTA-0, BTA-1, and BTA-2 usingthe [N-methyl-¹¹C]BTA-1 binding assay. The Ki's were: 3.0±0.8 nM forBTA-2; 9.6±1.8 nM for BTA-1; 100±16 nM for BTA-0; and 1900±510 nM forThT. Not only is the quaternary amine of ThT not necessary for bindingto Aβ fibrils, it appears to decrease binding affinity as well,

In Table 1 below are five different ¹¹C-labeled BTA derivatives wheretheir in vitro binding properties, log P values, and in vivo brainuptake and retention properties in mice have been determined.

TABLE 1 In vitro and in vivo properties of several promising ¹¹C-labeledThioflavin T derivatives. Ratio of K_(i) Mouse Brain Mouse Brain 2min/30 (nM) Uptake @ 2 Uptake @ 30 min Structure of ¹¹C-Labeled to Aβmin min Uptake BTA Compound fibrils logP (% ID/g*kg) (% ID/g*kg) Values

21 3.3(est.) 0.32 ± 0.07 0.17 ± 0.05 1.9

nottested 3.9(est.) 0.15 ± 0.06 0.16 ± 0.02 0.9

30 1.9(est.) 0.60 ± 0.04 0.39 ± 0.05 1.5

5.7 2.7 0.43 ± 0.11 0.094 ± 0.038 4.6

2.3 3.3(est.) 0.32 ± 0.09 0.42 ± 0.10 0.8

9.6 2.7 0.44 ± 0.14 0.057 ± 0.010 7.7

The data shown in Table 1 are remarkable, particularly for the¹¹C-labeled 6-MeO-BTA-1 and BTA-1 derivatives. These compounds displayedrelatively high affinity for Aβ, with Ki values <10 nM, and readilyentered mouse brain with uptake values >0.4% ID/g*kg (or >13% ID/g for30 g animals). Moreover, the 30 min brain radioactivity concentrationvalues were less than 0.1% ID/g*kg, resulting in 2 min-to-30 minconcentration ratios >4. Both of the N,N-dimethyl compounds cleared lessrapidly from mouse brain tissue than the N-methyl derivatives. Likewise,the only primary amine currently testable, 6-MeO-BTA-0, showed poorbrain clearance. This surprising and unexpected result supports thespecific use of the secondary amine (e.g. —NHCH₃) as in vivo imagingagent.

Example 7 In Vivo PET Imaging Experiments in Baboons

Large amounts of high specific activity (>2000 Ci/mmol) ¹¹C-labeledBTA-1,6-Me-BTA-1, and 6-MeO-BTA-1 were prepared for brain imagingstudies in 20-30 kg anesthetized baboons using the Siemens/CTI HR+tomograph in 3D data collection mode (nominal FWHM resolution 4.5 mm).Brain imaging studies were conducted following the intravenous injectionof 3-5 mCi of radiotracer. Typical attenuation- and decay-correctedtime-activity curves for a frontal cortex region of interest for each ofthe three compounds are shown in FIG. 6. It is noted that the absolutebrain uptake of these 3 compounds in baboons is very similar to that inmice (i.e., about 0.47 to 0.39% ID/g*kg). However, the normal brainclearance rate of all three radiotracers is considerably slower inbaboons compared to mice, with peak-to-60 min ratios in the range of 2.4to 1.6 compared to ratios as high as 7.7 at 30 min in mice. The rankorder of maximum brain uptake and clearance rate of the three compoundswere also the same in mice and baboons. Brain uptake of the radiotracersdid not appear to be blood flow-limited (FIG. 6, inset). Arterial bloodsamples in the baboons following the injection of all three compoundswere obtained, and showed that their metabolic profiles were quitesimilar. Only highly polar metabolites that eluted near the void volume(4 mL) of the reverse-phase analytical HPLC column were observed in theplasma at all time points following injection, while the unmetabolizedtracer eluted at about 20 mL. Typical amounts of unmetabolized injectatein plasma for all three compounds were about: 90% at 2 min; 35% at 30min; and 20% at 60 min.

Transverse PET images at two levels of baboon brain following the i.v.injection of 3 mCi of [N-methyl-¹¹C]BTA-1 are shown in FIG. 7. Theemission files collected 5-15 min post injection were summed to providethe images. Brain regions include: Ctx (cortex); Thl (thalamus); Occ(occipital cortex); and Cer (cerebellum). Note the uniform distributionof radioactivity throughout the brain, indicating lack of regionalbinding specificity in normal brain.

Example 8 Staining Amyloid Deposits in Post-Mortem AD and Tg Mouse Brain

Postmortem brain tissue sections from AD brain and an 8 month oldtransgenic PS1/APP [explain what this model is used to show] mouse werestained with unlabeled BTA-1. The PS1/APP mouse model combines two humangene mutations known to cause Alzheimer's disease in a doubly transgenicmouse which deposits Aβ fibrils in amyloid plaques in the brainbeginning as early as 3 months of age. Typical fluorescence micrographsare shown in FIG. 8, and the staining of amyloid plaques by BTA-1 inboth postmortem AD and PS1/APP brain tissue is clearly visible.Cerebrovascular amyloid also was brightly stained (FIG. 8, right). Theother characteristic neuropathological hallmark of AD brain,neurofibrillary tangles (NFT), are more faintly stained by BTA-1 in ADbrain (FIG. 8, left). NFT have not been observed in transgenic mousemodels of amyloid deposition.

Example 9 In Vivo Labeling and Detection of Amyloid Deposits inTransgenic Mice

Three 17 month-old PS1/APP transgenic mice were injectedintraperitoneally (ip) with a single dose of 10 mg/kg of BTA-1 in asolution of DMSO, propylene glycol, and pH 7.5 PBS (v/v/v 10/45/45).Twenty-four hours later, multiphoton fluorescence microscopy wasemployed to obtain high resolution images in the brains of living miceusing a cranial window technique. Typical in vivo images of BTA-1 in aliving PS1/APP mouse are shown in FIG. 9, and plaques andcerebrovascular amyloid are clearly distinguishable. The multiphotonmicroscopy studies demonstrate the in vivo specificity of BTA-1 for Aβin living PS1/APP transgenic mice.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification beconsidered as exemplary only, with the true scope and spirit of theinvention being indicated by the following claims.

As used herein and in the following claims, singular articles such as“a”, “an”, and “one” are intended to refer to singular or plural.

1. An amyloid binding compound having one of structures A-E or a watersoluble, non-toxic salt thereof:

wherein Z is S, NR′, O or CR′ in which case the correct tautomeric formof the heterocyclic ring becomes an indole in which R′ is H or a loweralkyl group:

wherein Y is NR¹R², OR², or SR²; wherein the nitrogen of

is not a quaternary amine; or an amyloid binding compound having one ofstructures F-J or a water soluble, non-toxic salt thereof:

wherein each Q is independently selected from one of the followingstructures:

wherein n=0, 1, 2, 3 or 4,

wherein Z is S, NR′, O, or C(R′)₂ in which R′ is H or a lower alkylgroup; wherein U is CR′ (in which R′ is H or a lower alkyl group) or N(except when U=N, then Q is not

wherein Y is NR¹R², OR², or SR²; wherein the nitrogen of

is not a quaternary amine; wherein each R¹ and R² independently isselected from the group consisting of H, a lower alkyl group,(CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X, CH₂—CH₂—CH₂X(wherein X=F, Cl, Br or I), (C═O)—R′, R_(ph), and (CH₂)_(n)R_(ph)(wherein n=1, 2, 3, or 4 and R_(ph) represents an unsubstituted orsubstituted phenyl group with the phenyl substituents being chosen fromany of the non-phenyl substituents defined below for R³-R¹⁴ and R′ is Hor a lower alkyl group); and wherein each R³-R¹⁴ independently isselected from the group consisting of H, F, Cl, Br, I, a lower alkylgroup, (CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X, O—CH₂—CH₂X,CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), CN, (C═O)—R′,N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, R_(ph),CR′═CR′-R_(ph), CR₂′-CR₂′-R_(ph) (wherein R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined for R¹-R¹⁴and wherein R′ is H or a lower alkyl group), a tri-alkyl tin and achelating group (with or without a chelated metal group) of the form W-Lor V—W-L, wherein V is selected from the group consisting of —COO—,—CO—, —CH₂O— and —CH₂NH—; W is —(CH₂)_(n) where n=0, 1, 2, 3, 4, or 5;and L is:

wherein M is selected from the group consisting of Tc and Re; or whereineach R¹ and R² is a chelating group (with or without a chelated metalgroup) of the form W-L, wherein W is —(CH₂)_(n) where n=2, 3, 4, or 5;and L is:

wherein M is selected from the group consisting of Tc and Re; or whereineach R¹-R¹⁴ independently is selected from the group consisting of achelating group (with or without a chelated metal ion) of the form W-Land V—W-L, wherein V is selected from the group consisting of —COO—, and—CO—; W is —(CH₂)_(n) where n=0, 1, 2, 3, 4, or 5; L is:

and wherein R¹⁵ independently is selected from the following:

or an amyloid binding, chelating compound (with or without a chelatedmetal group) or a water soluble, non-toxic salt thereof of the form:

wherein R¹⁵ independently is selected from the following:

and R¹⁶ is

wherein Q is independently selected from one of the followingstructures:

wherein n=0, 1, 2, 3 or 4,

wherein Z is S, NR′, O, or C(R′)₂ in which R′ is H or a lower alkylgroup; wherein U is N or CR′; wherein Y is NR¹R², OR², or SR²; whereineach R¹⁷-R²⁴ independently is selected from the group consisting of H,F, Cl, Br, I, a lower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or 3),CF₃, CH₂—CH₂X, O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (wherein X=F,Cl, Br or I), CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′,COOR′, R_(ph), CR′═CR′—R_(ph) and CR₂′-CR₂′-R_(ph) (wherein R_(ph)represents an unsubstituted or substituted phenyl group with the phenylsubstituents being chosen from any of the non-phenyl substituentsdefined for R¹⁷-R²⁰ and wherein R′ is H or a lower alkyl group).